CN102559854B - Standard plasmid molecular used for detecting transgenic soybean, corn and cotton and construction of the standard plasmid molecular - Google Patents
Standard plasmid molecular used for detecting transgenic soybean, corn and cotton and construction of the standard plasmid molecular Download PDFInfo
- Publication number
- CN102559854B CN102559854B CN201010595049.8A CN201010595049A CN102559854B CN 102559854 B CN102559854 B CN 102559854B CN 201010595049 A CN201010595049 A CN 201010595049A CN 102559854 B CN102559854 B CN 102559854B
- Authority
- CN
- China
- Prior art keywords
- strain
- gene
- corn
- transgenic
- standard plasmid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a necessary standard plasmid molecular and a construction method of the standard plasmid molecular in the field of transgenic crop detection. The construction method comprises the following steps: modifying an octo-gene plasmid molecular pTLE8 (Patent Application No.201010286884.3), adding a fusion fragment of 3'-transformation event specific sequences of transgenic corn Bt176 strain and Mon810 strain and corn internal reference gene Hmg specific sequence, and modifying into a deca-gene plasmid molecular pTLH10 (Lec1, 35S, NOS, PAT, Sad1, Cry1, Ab/c, Bt176, Mon810 and Hmg). The standard plasmid molecular constructed by the invention can completely replace a positive standard sample, and is suitable for screening transgenic soybean GTS40-3-2 strain, transgenic corn Bt176 and Mon810 strains and transgenic Bt cottons, and qualitative and quantitative PCR analysis and detection of gene specificity and strain specificity.
Description
Technical field
What the present invention relates to is a kind of plasmid molecule of technical field of molecular biology, specifically, relates to a kind of genetically engineered soybean, corn and cotton detection standard plasmid molecule and construction process thereof.
Background technology
Since transgenic plant commercialization, global transgenic plant cultivated area constantly expands, and 1996 to 2009 global transgenic crop cultivated areas add up to reach 6.9 hundred million hectares, and between more than 10 year, transgenic crop cultivated area increases 67 times.Genetically modified crops mainly genetically engineered soybean, transgenic corns and the transgene cotton of plantation.The cultivated area of genetically engineered soybean is maximum, 5,860 ten thousand hectares, accounts for 57% of global genetically modified crops cultivated area, is secondly transgenic corns (2,520 ten thousand hectares, account for 25%) and transgene cotton (1,340 ten thousand hectares, account for 13%).Along with the extensive plantation of genetically modified crops, its safety issue causes the most attention of the global public.In many countries particularly European Union, national legislation requires to carry out label explanation to transgenic product.
China has issued " agriculture GMO bio-safety management rules " May 23 calendar year 2001, on January 5th, 2002 discloses agriculture GMO bio-safety evaluation, mark and import security and manages two supporting management ways, determine the agriculture genetically modified organism catalogue that first implements identity management, and formal enforcement in 20 days March in 2002.
The enforcement of mark system depends on GMO detection technology.At present, the stdn detection method of also ununified in the world genetically modified crops and products thereof.The detection method of comprehensive various countries, mainly contains two types, namely based on exogenous gene expression product-protein detection and the DNA detection based on foreign gene insertion.For the exogenous DNA array inserted, PCR method is most widely used.PCR inspection policies can be divided into four kinds, and namely universal component selective mechanisms, gene specific detect, build specific detection and event-specific detection.Universal component screening PCR detects mainly gene fragment for the purpose of transgenosis universal component CaMV35S promotor and NOS terminator; It is that the DNA fragment specific inserting foreign gene detects fragment as object that gene specific PCR detects; It is realized by the joining region sequence detecting exogenous insertion vector and Plant Genome that strain specificity PCR detects.Due to each Transgenic Plant Lines, all there is the joining region sequence of special exogenous insertion vector and Plant Genome, and joining region sequence is single copy, so event-specific detection method has higher specificity and accuracy.Event-specific detection has become the emphasis of current detection GMOs research, and step by step by international each testing laboratory is adopted.
When applying PCR and detecting transgenic product, needing, positive control is set detected activity is monitored.Usually using the genomic dna extracted from positive criteria product (CRMs) as positive control, but because genetically modified crops exist biological safety and the restriction with prior art, genetically modified crops positive criteria product are difficult to obtain, therefore development of new positive control material is needed badly, to meet the detection GMOs demand of development.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of genetically engineered soybean, corn and cotton detection standard plasmid molecule and construction process thereof are provided, make it that positive criteria material can be replaced to detect for the quantitative and qualitative analysis PCR of genetically engineered soybean, corn and cotton.Utilize the PCR of 3 ' end transformation event of the principle design of fusion DNA vaccine technology corn Hmg gene, transgenic corns Bt176 and Mon810 strain to merge primer, obtain digenic fusion fragment through fusion DNA vaccine amplification.Utilize molecular cloning technology fusion fragment to be inserted into plasmid molecule pTLE8 (number of patent application 201010286884.3), obtain group plasmid molecule pTLH10.The standard plasmid molecule pTLH10 that the present invention builds can as the positive control in testing process, and the difficult problem detecting Plays material want for solving genetically modified crops provides an effective way and effectively can ensure the carrying out of testing.
The present invention is realized by following experimental program, standard plasmid molecule of the present invention, with alternative transgenic soybean lines GTS40-3-2 and turn PAT soybean, transgenic corns Bt176 and Mon810 strain, transgenosis Bt are cotton, detect for quantitative and qualitative analysis PCR.This standard plasmid molecule contains soybean reference gene Lectin1, 35S promoter, NOS terminator, pat gene, 5 ' end transformation event sequence of genetically engineered soybean GTS40-3-2 strain, cotton native gene Sad specific fragment, the specific fragment of the foreign gene CrylAb/c fusion gene of transgenic Bt cotton, corn reference gene Hmg specific fragment, the partial sequence of 3 ' end transformation event of transgenic corns Bt176 strain and 3 end transformation event specific fragments of Mon810 strain.
A kind of genetically engineered soybean of the present invention, corn and cotton detection standard plasmid molecule and construction process thereof, comprise the following steps:
(1) utilize GcnBank database lookup and obtain corn reference gene Hmg, 3 ' end transformation event of transgenic corns Bt176 strain and 3 ' end transformation event specific sequence of Mon810 strain;
(2) utilize Primer Premier 5.0 software design PCR Auele Specific Primer and carry out blast checking;
(3) increase above-mentioned three genes respectively;
(4) adopt fusion DNA vaccine method that three gene splicings are become a fragment;
The first round reacts: with the genomic dna of transgenic corns Bt176 and Mon810 for template, carry out pcr amplification with primer corresponding in claim 3; Reaction system is: cumulative volume is 50 μ l, wherein 10 times of Taq damping fluid 5 μ l, dNTPs (10mM) 4 μ l, each 2 μ l of upstream and downstream primer (10 μMs), and template (100ng/ μ l) 1 μ l, uses ddH
2o complements to 50 μ l.
Response procedures is: 95 DEG C of 5min; 30 circulations (94 DEG C of 30s, 58.2-59.6 DEG C of 30s, 72 DEG C of 60s); 72 DEG C of 10min; 4 DEG C of preservations; Product Labeling is P
hmg, P
bt, P
mon;
Second takes turns reaction, increases in two steps:
The first step: get each 70ng of the first round anti-product, 10 times of Taq damping fluid 5 μ l, dNTPs (10mM) 4 μ l, uses ddH
2o complements to 50 μ l; Make P
hmg, P
bt, P
monbe connected;
Response procedures: 11 circulations (94 DEG C of 15s, 60 DEG C of 20s, 72 DEG C of 10min), 4 DEG C of preservations;
Second step: add each 1 μ l of primer Bt-P1 and Hmg-P2 in the first step reaction solution respectively;
Response procedures:: 94 DEG C of 3min, 28 circulations (94 DEG C of 15s, 60 DEG C of 1min, 72 DEG C of 2min), 4 DEG C of preservations; P is labeled as by after fusion DNA vaccine product rubber tapping purifying
bH3.
(5) because receiving purifying P
bH13, be connected to pMD
tM19-TSimple carrier, proceeds to intestinal bacteria TOP10 screening and obtains middle plasmid molecule pTBH3.
(6) plasmid molecule pTLE8 and pTBH3 is carried out double digestion with restriction enzyme EcoR I and XbaI respectively, reclaim this two kinds of digestion products, connect with T4DNA ligase enzyme and obtain plasmid molecule pTLH10.
(7) the quantitative PCR detecting method checking of standard plasmid molecule pTLH10.
Described quantitative PCR checking, refer to quantitative detection limit and the characteristic such as repeatability and repeatability of corn native gene HmgI and transgenic corns Bt176, Mon810 strain transformation event specific sequence in the standard plasmid molecule pTLH10 built with quantifying PCR method analysis, to identify that this standard plasmid molecule replaces the ability of positive criteria material
Beneficial outcomes of the present invention is, utilizes fusion DNA vaccine, enzyme is cut, connects equimolecular clone technology and construct standard plasmid molecule pTLH10 containing ten genes.This standard plasmid molecule can replace positive criteria material for the quantitative PCR detection of genetically engineered soybean, corn and cotton, well solve the problem of the positives mark material want of testing process, without the need to providing multiple positive gene group DNA as positive control in testing process; This standard plasmid is divided and has proceeded to intestinal bacteria, its stable preservation can have been grown; Also new exogenous genetic fragment can be added on the basis of original standard plasmid molecule, to meet the needs of increasing genetically modified crops material tests.This standard plasmid molecule pTLH10 has higher specificity and repeatability, sensitivity advantages of higher in reality detects, apply this standard plasmid molecule when carrying out actual sample quantitative analysis, in the allowed band that the deviation of sample detection result is extensively approved in the world.Therefore, the standard plasmid molecule pTLH10 built in the present invention is applicable to the quantitative analysis to the gm content in genetically engineered soybean, corn and cotton and converted products thereof completely.
Accompanying drawing explanation
Accompanying drawing is the sequencing result of ten gene standard plasmid molecule pTLH10
Italic overstriking be fusion DNA vaccine primer, between ten genes, introduce four restriction enzyme NotI, SalI, EcoR I and Xba I.Be primer and the probe sequence of quantitative PCR in square frame.
Embodiment
In order to explain enforcement of the present invention more fully, what provide genetically engineered soybean, corn and cotton standard molecule contrast prepares embodiment.These embodiments are only explain instead of restriction point scope of invention.The experimental technique of tool condition is not indicated, usually conveniently conditional operation in the following example
Embodiment 1: the structure of standard plasmid molecule
One, experiment material
Transgenic corns Bt176 strain and Mon810 strain; Non-transgenic corn kind.
Two, experiment reagent
PMD
tM19-T Simple Vector is purchased from Dalian Bao Bio-Engineering Company; DNTPs, Taq archaeal dna polymerase, DNA marker I, II and marker III are purchased from Beijing Quanshijin Biotechnology Co., Ltd; Restriction enzyme Not I, Sal I, EcoR I, Xba I are purchased from NEB Beijing company limited; The plasmid Mini Kit that plasmon DNA extraction and purifying adopt Beijing Tian Gen biochemical technology company limited to develop; Other biochemical reagents are import packing or domestic analytical pure.
Three, laboratory apparatus
TC-512 type PCR amplification instrument (TECHNE company of Britain); Geliance 200DNA running gel imager (PerkinElmer company of the U.S.); Nano-Drop ND-1000 nucleic acid-protein instrument (Nano Drop company of the U.S.); Whizzer; Thermostat water bath; Incubator; It equality.
Four, test method and process
1, plant genome DNA extracts-SDS method
A, take the corn sample that 0.1g pulverizes through pulverizer, add the 2%SDS extract of 1ml 65 DEG C of preheatings, 10 μ lRNase-A mix, and in 65 DEG C of incubation 30min, are placed in the centrifugal 10min of 13000r after room temperature.
B, get the KAc resetting and adding 0.6 times of volume, after ice bath 20min, the centrifugal 10min of 13000r, gets and resets and add 2 times of volume precooling dehydrated alcohols, mixing.
C, in-20 DEG C of centrifugal 10min of standing 30min, 13000r, fill with clearly, precipitation uses 70% washing with alcohol, abandons supernatant, repeat once after the short period of time is centrifugal.The molten precipitation of 200 μ l TE is added, 20 DEG C of preservations after seasoning.
D, get 2 μ l extract DNA sample, with 1% agarose gel electrophoresis detection DNA quality.The DNA concentration and purity that utilize nucleic acid instrument to measure to extract.
2, design of primers
3 ' end transformation event specific sequence of corn native gene Hmg, transgenic corns Bt176 strain and Mon810 strain is searched in GenBank, utilize software Primer5.0 to design 3 pairs of qualitative PCR primers, be respectively used to 3 ' end transformation event specific sequence of amplification corn reference gene specific sequence Hmg, transgenic corns Bt176 strain and Mon810 strain.Concrete primer sequence is in table 1.
Table 1 builds the PCR primer sequence of standard plasmid molecule
3, trigenic independent pcr amplification
According to the primer of table 1, with transgenic corns genomic dna for template, respectively pcr amplification is carried out to 3 ' end transformation event specific sequence of corn native gene Hmg, transgenic corns Bt176 strain and Mong10 strain.
Conveniently PCR method carries out the independent amplification of each object fragment.Amplification uses Taq archaeal dna polymerase, and reaction conditions and the amplified production length of each fragment amplification are as shown in table 2.The amplified production agarose gel electrophoresis of 1.5% detects, and determines object band, cuts glue Gel ExtractionMiniKit and reclaim.Reduce the add-on of EB in gel preparation course, require EB concentration generally lower than 0.15 μ g/ml.
The reaction conditions of table 2 fragment amplification to be fused and amplified production length
4, fusion DNA vaccine reaction
Measure each because receiving object fragment concentrations in product, equimolar fragment to be fused (amount of each gene to be fused is 70ng) is added in 50 μ l systems, do not add primer, the complementation using Taq archaeal dna polymerase to carry out each fragment extends, 95 DEG C of 15s, 60 DEG C of 20s, 72 DEG C of 10min, 11 circulations.Outside primer and Bt-P1 (10 μMs) and each 4 μ l of Hmg-P2 (10 μMs) is added, response procedures: 94 DEG C of 30s, 28 circulations (94 DEG C of 30s, 60 DEG C of 1min, 72 DEG C of 2min), 4 DEG C of preservations in reaction solution; The amplified production agarose gel electrophoresis of 1.5% detects, and determines object band, cuts glue Gel Extraction MiniKit and reclaim.
5, the structure of pTLH10 plasmid molecule
Utilize the method for molecular cloning that trigenic fusion fragment is reclaimed product and be connected to pMD
tMon 19-T Simple carrier, transformation of E. coli TOP10 competent cell after 42 DEG C of heat shock 90s, blue hickie screens positive bacterium colony and carries out bacterium colony PCR qualification, the positive colony order-checking that choosing qualification is correct.By extract after positive colony bacterium expanding propagation its plasmid pTBH3,
Plasmid molecule pTBH3 and pTLE8 is carried out double digestion with EcoR I and Xba I respectively, after agarose gel electrophoresis, reclaims goal gene fragment BH3 and carrier segments pTLE7 respectively, connect with T4DNA ligase enzyme and obtain plasmid molecule pTLH10.Reaction system is in table 3, and 16 DEG C connect after 30min, 4 DEG C of connections of spending the night, will connect product conversion TOP10 competence bacterial strain.Utilize bacterium colony PCR to identify recombinant bacterial strain, extract plasmid pTLH10 according to plasmid extraction kit specification sheets.
Table 3 builds the ligation system of plasmid molecule pTLH10
6, pTLH10 standard plasmid molecule linearizing
The pTLH10 plasmid restriction endonuclease Sal I built or EcoR I carries out linearizing, as the standard molecule of transgenic corns quantitative analysis.Gel reclaims linearizing plasmid DNA, with nucleic acid instrument measure linear plasmid DNA concentration.According to plasmid molecule size, the total mass number of plasmid can be converted into copy number.
Five, experimental result
1, trigenic independent pcr amplification
Conveniently PCR method carries out the independent amplification of each object fragment of Bt176, Mong10 and Hmg, obtains 372bp, 365bp and 354bp fragment respectively, is consistent with expection size.
2, fusion DNA vaccine reaction
Bt176, Mon810 and Hmg are merged by bridging PCR, called after BH3, size is 1011bp, consistent with expection size.
3, the structure of pTLH10 standard plasmid molecule and sequence verification
Upper 10 standard plasmid molecules of plasmid molecule pTL built are through sequence verification, containing Lectin 1, 35S, NOS, PAT, 5 ' end transformation event of genetically engineered soybean GTS40-3-2 strain, cotton native gene Sad specific fragment, the specific fragment of the external source CrylAb/c fusion gene of transgenic Bt cotton, corn reference gene Hmg specific fragment, the partial sequence of 3 ' end transformation event of transgenic corns Bt176 strain and 3 ' end transformation event specific fragment of Mon810 strain, totally ten genes, total length is 3360bp, the homology of each gene order that ten genes log in GenBank is respectively 100%.
Embodiment 2: the application of standard plasmid molecule in reality detects of structure
One, enzyme and reagent
Real-time fluorescence quantitative PCR detection kit (Premix Ex Taq
tM(Perfect Real Time)) purchased from precious biotechnology (Dalian) company limited; Primer and TaqMan probe are synthesized by Jikang Biotechnology Co Ltd, Shanghai; Other biochemical reagents are import packing or domestic analytical pure.
Two, laboratory apparatus
Real-time fluorescence quantitative PCR instrument 7500 (ABI company).
Three, experimental technique and process
1, fluorescence quantification PCR primer and probe design synthesis
According to Hmg and Bt176 in standard plasmid molecule and Mon810 strain 3 ' holds transformation event sequence, adopts software Primer Premier 5.0 to design quantification PCR primer and probe, in table 4.5 ' end mark reporter fluorescence dyestuff FAM of TaqMan probe, 3 ' end mark cancellation non-fluorescence dyestuff BHQ.Primer and probe synthesize by Jikang Biotechnology Co Ltd, Shanghai.Quantitative fluorescent PCR reaction system and reaction conditions are in table 5.
The quantitative primer of table 4Lectin I and EPSPS gene and probe
Table 5 quantitative PCR reaction system and reaction conditions
2, the foundation of the border directrix curve of transgenosis Bt176 and Mon810 strain
Using linearization plasmid pTLH10 as standard DNA sample, be diluted to 4.85 × 10
5copy/μ l, 4.85 × 10
4copy/μ l, 4.85 × 10
3copy/μ l, 4.85 × 10
2copy/μ l and 4.85 × 10
1copy/μ l, production standard curve.
3, standard plasmid molecule pTLH10 is used for the limit of detection of quantitative PCR
Using the standard plasmid molecule DNA sample of different concns (as 48.5 copy/μ l, 20 copy/μ l, 10 copy/μ l, 6 copy/μ l) as LOD and LOQ of sample determination quantitative PCR detecting method.Each reaction repeats secondary, according to the linear relationship between the typical curve of quantitative pcr amplification and amplification fluorescent signal, when determining to utilize the standard plasmid molecule pTLH10 replacement positive criteria material built, the limit of detection of transgenosis Bt176 and Mon810 strain specificity quantitative PCR detecting method.
4, the repeatability of standard plasmid molecule pTLH10 quantitative PCR detection system and repeatability
Optimize 3 ' end transformation event specific quantification PCR detection system and reaction conditions of transgenic corns Bt176 and Mon810 strain, respectively with the standard plasmid molecule genome DNA sample of different concns (as 485 × 10
5copy/μ l, 4.85 × 10
4copy/μ l, 4.85 × 10
3copy/μ l, 4.85 × 10
2copy/μ l, 4.85 × 10
1copy/μ l) carry out repeatability and circulation ratio test, each reaction repeats secondary.According to the typical curve of quantitative pcr amplification, determine repeatability and the repeatability of 3 ' end transformation event specific quantification PCR reaction of transgenic corns Bt176 and the Mon810 strain set up according to standard plasmid molecule pTLH10.
Four, experimental result
1, the foundation of typical curve
Set up quantitative analysis method using linearization plasmid DNA as standard substance, the amplification efficiency of the typical curve of Hmg and Bt176 is respectively 99.581% and 99.889%, and relation conefficient is respectively 0.996 and 0.997; The amplification efficiency of the typical curve of Hmg and Mon810 is respectively 93.266% and 93.324%, and relation conefficient is respectively 0.998 and 0998, illustrates to have good Ct value-starting point concentration dependency, can realize carrying out accurate quantitative analysis research to corn sample.
2, the mensuration of quantitative detection limit
Utilize the quantitative PCR reaction system optimized, respectively using the standard plasmid DNA sample of different concns (48.5 copy/μ l, 20 copy/μ l, 10 copy/μ l, 6 copy/μ l) as unknown sample, repeat experiment by 3 times, the quantitative detection limit of the quantitative PCR of Bt176 and Mon810 is respectively 10,20 copies.
3, quantitation curves repeatability is analyzed
Carry out reperformance test using the standard plasmid molecule DNA of different copy number as standard substance respectively, do three repetitions in the same way with condition, each three parallel.Repeatability result is as shown in table 6,7.The phase standard deviation of the Ct value obtained between Bt176 parallel reactor and between different repetition is less than 5.83%, the relative standard deviation of Mon810 is less than 2.98% (can accept within standard-required 35%), illustrate that the repeatability that quantitative PCR reacts is fine, there is very high stability, may be used for the further quantitative analysis of actual sample.
The repeatability analysis of table 6Bt176 quantitation curves
The repeatability analysis of table 7Mon810 quantitation curves
Claims (1)
1. a genetically engineered soybean, corn and cotton detection standard plasmid molecule, it is characterized in that, containing soybean reference gene Lectin 1, 35S promoter, NOS terminator, pat gene, 5 ' end transformation event 35SG of genetically engineered soybean GTS40-3-2 strain, cotton native gene Sad specific fragment, the specific fragment of the foreign gene Cry1Ab/c fusion gene of transgenic Bt cotton, corn reference gene Hmg specific fragment, the partial sequence of 3 ' end transformation event of transgenic corns Bt176 strain and 3 ' end transformation event specific fragment of Mon810 strain, its sequencing is Lectin 1+35S+NOS+PAT+35SG+Sad+Cry1Ab/c+Bt176+Mon810+Hmg, wherein 3 ' end transformation event of transgenic corns Bt176 strain, 3 ' end transformation event of Mon810 strain and the primer sequence of corn reference gene Hmg specific sequence see the following form
。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010595049.8A CN102559854B (en) | 2010-12-20 | 2010-12-20 | Standard plasmid molecular used for detecting transgenic soybean, corn and cotton and construction of the standard plasmid molecular |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010595049.8A CN102559854B (en) | 2010-12-20 | 2010-12-20 | Standard plasmid molecular used for detecting transgenic soybean, corn and cotton and construction of the standard plasmid molecular |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102559854A CN102559854A (en) | 2012-07-11 |
| CN102559854B true CN102559854B (en) | 2015-05-20 |
Family
ID=46406458
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010595049.8A Active CN102559854B (en) | 2010-12-20 | 2010-12-20 | Standard plasmid molecular used for detecting transgenic soybean, corn and cotton and construction of the standard plasmid molecular |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102559854B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651511B (en) * | 2015-02-14 | 2016-07-27 | 中国农业科学院油料作物研究所 | A kind of positive plasmid molecule pBI121-Screening and application thereof |
| CN104762310A (en) * | 2015-04-02 | 2015-07-08 | 贵州省烟草科学研究院 | High-coverage standard reference plasmid for detecting transgenic tobacco and application of high-coverage standard reference plasmid for detecting transgenic tobacco |
| CN106244674A (en) * | 2016-06-07 | 2016-12-21 | 谱尼测试集团深圳有限公司 | A kind of standard plasmid molecule for transgenic wheat detection and construction method thereof |
| CN111826388A (en) * | 2020-07-07 | 2020-10-27 | 黑龙江省农业科学院农产品质量安全研究所 | Unauthorized transgenic corn screening positive plasmid molecule pYMSC-1905 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101063171A (en) * | 2007-05-24 | 2007-10-31 | 上海交通大学 | Standard plasmid molecule for detection of genetic improved soybean strain GTS40-3-2 and constructing method thereof |
| CN101724702A (en) * | 2009-12-23 | 2010-06-09 | 天津市农业科学院中心实验室 | Standard molecule contrast for genetically modified maize detection and prepration method thereof |
| CN101775440A (en) * | 2010-02-05 | 2010-07-14 | 吉林省农业科学院 | Plasmid control molecule for detection of transgenic soybean and building method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2140266A1 (en) * | 2007-03-26 | 2010-01-06 | Toxispot A/S | Quantification of analyte molecules using multiple reference molecules and correlation functions |
-
2010
- 2010-12-20 CN CN201010595049.8A patent/CN102559854B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101063171A (en) * | 2007-05-24 | 2007-10-31 | 上海交通大学 | Standard plasmid molecule for detection of genetic improved soybean strain GTS40-3-2 and constructing method thereof |
| CN101724702A (en) * | 2009-12-23 | 2010-06-09 | 天津市农业科学院中心实验室 | Standard molecule contrast for genetically modified maize detection and prepration method thereof |
| CN101775440A (en) * | 2010-02-05 | 2010-07-14 | 吉林省农业科学院 | Plasmid control molecule for detection of transgenic soybean and building method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 曹际娟.建立转基因作物及产品外援抗性基因检测技术体系的研究.《中国优秀硕博士学位论文全文数据库(博士)》.2004,第33、34、40、57-61、76-77页. * |
| 转基因玉米特异性检测阳性标准分子的构建与应用;沈雨萌等;《玉米科学》;20100430;第18卷(第4期);第52-57页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102559854A (en) | 2012-07-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110777220B (en) | Primer group, probe, RPA test strip kit and identification method | |
| CN102559854B (en) | Standard plasmid molecular used for detecting transgenic soybean, corn and cotton and construction of the standard plasmid molecular | |
| CN102162012B (en) | Qualitative PCR detection method for transgenic rice kefeng No. 6 | |
| Wang et al. | Construction of a reference plasmid molecule containing eight targets for the detection of genetically modified crops | |
| CN101824411B (en) | Flanking sequence of transgenic rice Kefeng No. 6 and qualitative PCR detection method | |
| CN104032007A (en) | Method for detecting exogenous gene copy number in transgenic tobacco | |
| CN102409082B (en) | Five-gene standard plasmid molecule for transgenic soybean detection and construction thereof | |
| CN103215346A (en) | Recombinant standard plasmid and kit used in transgenic rice screening | |
| Wu et al. | Development and application of a general plasmid reference material for GMO screening | |
| CN102409081B (en) | A kind of be used for genetically engineered soybean and transgene cotton detection standard plasmid molecule and construction process thereof | |
| CN104988236A (en) | Transgene wheat B73-6-1 line specificity quantitative PCR detection kit and applications thereof | |
| CN103146824A (en) | Recombinant standard plasmid and kit for PCR (Polymerase Chain Reaction) detection of transgenic rice | |
| CN102134602A (en) | Primer, probe, test kit and method for testing Xa21 gene modified rice or products thereof | |
| AU2002212678B8 (en) | Methods of quantitative detection of genetic recombinants and standard molecules for the methods. | |
| CN102286624B (en) | Qualitative and quantitative PCR (polymerase chain reaction) detection method for strain specificity of transgenic carnation Moonlite | |
| CN112280906B (en) | DPO-PCR primer pair for detecting arabis mosaic virus and bean pod mottle virus and application thereof | |
| CN102492777B (en) | Standard plasmid molecule for transgenic maize Mon810 detection and construction method thereof | |
| CN106591340B (en) | Standard plasmid molecule for qualitative detection of transgenic organism and product gene thereof | |
| CN102586303B (en) | Standard plasmid of transgenic Bt rice and application of standard plasmid | |
| CN102409080A (en) | Two-gene standard plasmid molecule used for detecting genetically modified soybeans and building method thereof | |
| CN104561313A (en) | Endogenous reference gene suitable for detection of sesame exogenous gene copy number and building method and application of endogenous reference gene | |
| CN112646829B (en) | Plasmid standard molecule capable of being used for detecting multiple crops and multiple exogenous genes | |
| CN102199599B (en) | Specific detection target sequences of transgenic tomato, plasmid molecule and its detection kit thereof | |
| CN102586304B (en) | Standard plasmid containing various transposable elements for insect-resistant rice and cotton and application of standard plasmid | |
| CN102586302A (en) | Standard plasmid of transgenic Bt cotton and application of standard plasmid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |